The present invention relates to the technology for determining the base sequences of DNA and RNA, which are the materials that carry the genetic information in life forms, particularly to the technology for determining base sequences by hybridization.
The hybridization method that uses for the probe a single-stranded nucleic acid polymer complementary to the sequence of a region of the target nucleic acid polymer is known as a method of detecting a specific genetic sequence in nucleic acid polymers such as DNA or determining whether two nucleic acid polymers are identical.
By the hybridization method, a target nucleic acid polymer is immobilized to a support such as a glass slide or silicon chip, and an aqueous solution containing a probe nucleic acid polymer labeled with a radioisotope (RI) or fluorescent substance is placed on the film. If the probes hybridize to the target nucleic acid polymer, only the hybridized probes remain on the film after washing. The presence of a specific sequence in the target nucleic acid polymer can be determined by detecting the radiation or fluorescence from the IR or fluorescent substance label of the probes or the color of the precipitate.
The radioactive labeling method requires a considerable cost and labor for the facility for handling radioactive substances and its management. The method also has a health problem of people who handle radioactive substances. For this reason, the method that uses fluorescent substances for labeling and performs detection using a fluorescence detection system is in common use grin recent years.
However, a special modification treatment which takes a lot of time and labor is needed to label nucleic acid polymer probes with a fluorescent substance. Moreover, the fading of the fluorescent substance inevitably occurs because of irradiation of the exciting light, and hence an expensive dedicated fluorescence detection system is needed for a high-accuracy measurement.
Further, the immobilization efficiency of a target nucleic acid polymer is very low, and a large amount of a target nucleic acid polymer is needed for immobilization. Therefore, fluorescence detection is difficult when only a small amount of target nucleic acid polymers can be obtained.
There is another method that immobilizes single-stranded nucleic acid polymer probes directly to a solid support, hybridizes a target nucleic acid polymer with the probes, and performs the detection using the Plasmon Resonance method or Quartz Oscillator method. This method of detecting base sequences also has problems that a modification of nucleic acid polymers is required to immobilize them to the support and that the control of immobilization is difficult.
The present invention was made under the situation described above. The object of the present invention is therefore to provide an improved substrate for detecting base sequences and method of detecting base sequences using the substrate which can control the immobilization efficiency of a probe or target nucleic acid polymer to the substrate at a best condition for hybridization and consequently can detect the base sequence of a target nucleic acid polymer from a very small amount of the target nucleic acid polymer without need of leveling nucleic acid polymers with isotopes or fluorescent substances and without need of modifying the molecular structure of nucleic acid polymers.
The substrate for detecting base sequences of the present invention comprises a transparent support, a thin metal film formed on one side of the transparent support, and a self-assembled monolayer with a nonionic aromatic compound (such as anthracene, pyrene, naphthalene, phenanthrene and naphthene) being an intercalator of nucleic acid polymers dispersed over the surface formed on the metal film.
The method of manufacturing the substrate for detecting base sequences comprises the following steps: synthesizing a compound (such as a disulfide compound and thiol compound) containing a nonionic aromatic compound that is an intercalator of nucleic acid polymers; and immersing a transparent support with a thin metal film formed on one side in a solution containing the compound to let the self-assembled monolayer with the nonionic aromatic compound dispersed over the surface formed on the metal film.
The method of manufacturing the substrate for detecting base sequences that has a self-assembled monolayer with anthracene dispersed over the surface comprises the following steps: synthesizing a disulfide compound containing anthracene for the intercalator of nucleic acid polymers; and immersing a transparent support with a thin metal film formed on one side in a solution containing the compound to let a self-assembled monolayer of disulfide with anthracene dispersed over the surface form on the surface of the support.
The method of manufacturing the substrate above for detecting base sequences that has a self-assembled monolayer with pyrene dispersed over the surface comprises the following steps: synthesizing a thiol compound containing pyrene for the intercalator of nucleic acid polymers; and immersing a transparent support with a thin metal film formed on one side in a solution containing the compound to let a self-assembled monolayer of thiol with pyrene dispersed over the surface form on the surface of the support.
The method of detecting base sequences using the substrate comprises the following steps: letting the self-assembled monolayer adsorb a nucleic acid polymer (probe or target) to immobilize the nucleic acid polymer to the substrate; and performing the hybridization of another nucleic acid polymer (target or probe) and the nucleic acid polymer immobilized to the substrate on the substrate.